Haemophilus ducreyi is the causative agent of chancroid, a sexually transmitted genital ulcer disease that facilitates transmission and acquisition of human immunodeficiency virus (HIV) and thus contributes to the spread of HIV in areas with endemic chancroid. H. ducreyi resides primarily in the skin of infected individuals, where it is surrounded by phagocytes but remains extracellular. A key component of the innate immune response to pathogens is production of bactericidal antimicrobial peptides (APs). During bacterial infection of human skin, resident keratinocytes and infiltrating phagocytes secrete several kinds of APs, including a- defensins, [unreadable]-defensins, and cathelicidin LL37, into the extracellular milieu. Resistance to killing by APs is an important virulence mechanism of human pathogens that has not been studied in H. ducreyi. The long-term goal of this project is to understand mechanisms H. ducreyi uses to resist being killed by APs. Our preliminary studies showed that at least one class of APs, the ?-defensins, are present in natural chancroidal ulcers. To examine susceptibility of H. ducreyi to human APs, we developed an AP bactericidal assay. In this assay, H. ducreyi resisted killing by several APs that the organism should encounter during human infection. The AP resistance phenotype was similar in the two known classes of H. ducreyi strains. In vivo expression studies with specimens from the human model of H. ducreyi infection demonstrated that H. ducreyi expresses two transport systems during infection that confer resistance to APs in other bacterial pathogens. One expressed transporter is a Sap (sensitive to antimicrobial peptides) influx pump, and the other is an MTR (multiple transferable resistance) efflux pump. We hypothesize that the Sap and MTR transport systems contribute to AP resistance and virulence of H. ducreyi. In Aim 1 of the proposal, we will generate isogenic null mutants in one gene of each transport system and test the effects of the mutations on susceptibility to APs. To examine potential additive effects of expressing both Sap and MTR, we will also generate a double mutant lacking expression of both pumps and test its susceptibility to AP-mediated killing. In Aim 2, we will test the role of Sap and MTR in virulence during human infection by testing the mutants generated in Aim 1, alongside the parent strain, in the human model of H. ducreyi infection. APs are currently being studied for their utility in therapies such as topical microbicides designed to prevent sexually transmitted infections including chancroid. The work proposed here represents the first step in understanding how H. ducreyi interacts with and resists killing by APs and will provide important information for development of preventative therapies that include APs. [unreadable] [unreadable] Haemophilus ducreyi, which causes chancroid, is an important pathogen because of its ability to facilitate HIV transmission and its strong correlation with the spread of HIV in areas with endemic chancroid. The work in this application will define genes involved in the resistance of H. ducreyi to killing by human antimicrobial peptides and will determine the roles of these genes in virulence during human infection. Antimicrobial peptides, which kill many pathogens, are being studied for their utility in preventative therapies targeting sexually transmitted infections; the information learned from these studies will be important for developing such therapies. [unreadable] [unreadable] [unreadable] [unreadable]